TECHNICAL FIELD
[0001] The present invention relates to the technical field of biomedicine or biopharmaceuticals,
and specifically, relates to an antibody or an antigen-binding fragment thereof, or
a chimeric antigen receptor that targets Claudin18.2, as well as a preparation method
and use of the same for preparing a pharmaceutical composition, treating, preventing,
detecting or diagnosing a disease.
BACKGROUND
[0002] Claudin18.2 is transiently expressed in gastric epithelial cells only, and is seldom
expressed in other normal tissues. However, the expression of Claudin18.2 is abnormally
elevated in many cancerous tissues (
Niimi, Mol. Cell Biol., 21:7380-90, 2001). Claudin18.2 is expressed in gastric cancer, esophageal cancer, pancreatic cancer,
lung cancer, ovarian cancer and other tumors. Antibodies targeting Claudin18.2 can
mediate specific lysis of tumor cells through ADCC, CDC, inducing apoptosis and direct
inhibition of proliferation. Therefore, Claudin18.2 is currently the most promising
target in treatment of gastric cancer, esophageal cancer, pancreatic cancer, lung
cancer and ovarian cancer.
[0003] Claudin18.1 is selectively expressed in epithelial cells of normal lungs and stomachs.
The presence of two different variants introduces more complexity to Claudin18 molecules
(
Niimi, Mol. Cell Biol., 21:7380-90, 2001). How to further improve the effectiveness and safety is an issue to be considered
in the field.
[0004] IMAB362 developed by Ganymed (Chinese Patent No.
CN201380026898.3) is one of the first Claudin18.2 antibodies put into clinical trials. In its Phase
II clinical trials for gastric cancer, the antibody used in combination with chemotherapy
significantly prolonged the survival (13.2 vs. 8.4 months) compared with standard
chemotherapy, and had more significant efficacy and a longer median survival time
(16.7 months) in patients with high expression of Claudin18.2 (NCT01630083).
[0005] The present invention provides an antibody or an antigen-binding fragment thereof,
or a chimeric antigen receptor T cell that targets Claudin18.2 with good efficacy
for Claudin18.2-positive tumors, bringing new hope to patients with advanced gastric
cancer, pancreatic cancer, and the like.
SUMMARY
[0006] All embodiments concerning VL (light chain variable region), VH (heavy chain variable
region), LCDR (light chain complementarity determining region), HCDR (heavy chain
complementarity determining region), LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3
throughout the present invention may be implemented alone or in any combination.
[0007] In one aspect, the present invention provides an antibody or an antigen-binding fragment
thereof, wherein:
in some embodiments, the antibody or the antigen-binding fragment thereof comprises
any of the following combinations:
- (1) three light chain complementarity determining regions comprising an LCDR1 amino
acid sequence set forth in SEQ ID NO: 5, an LCDR2 amino acid sequence set forth in
SEQ ID NO: 6 and an LCDR3 amino acid sequence set forth in SEQ ID NO: 7, and/or three
heavy chain complementarity determining regions comprising an HCDR1 amino acid sequence
set forth in SEQ ID NO: 8, an HCDR2 amino acid sequence set forth in SEQ ID NO: 9
and an HCDR3 amino acid sequence set forth in SEQ ID NO: 10;
- (2) three light chain complementarity determining regions comprising an LCDR1 amino
acid sequence set forth in SEQ ID NO: 11, an LCDR2 amino acid sequence set forth in
SEQ ID NO: 12 and an LCDR3 amino acid sequence set forth in SEQ ID NO: 13, and/or
three heavy chain complementarity determining regions comprising an HCDR1 amino acid
sequence set forth in SEQ ID NO: 14, an HCDR2 amino acid sequence set forth in SEQ
ID NO: 15 and an HCDR3 amino acid sequence set forth in SEQ ID NO: 16;
in some embodiments, the antibody or the antigen-binding fragment thereof comprises
any of the following combinations:
- (1) a light chain variable region of an amino acid sequence set forth in SEQ ID NO:
1 and/or a heavy chain variable region of an amino acid sequence set forth in SEQ
ID NO: 2;
- (2) a light chain variable region of an amino acid sequence set forth in SEQ ID NO:
3 and/or a heavy chain variable region of an amino acid sequence set forth in SEQ
ID NO: 4.
[0008] In one aspect of the present invention, the antibody or the antigen-binding fragment
thereof includes monoclonal antibodies, polyclonal antibodies, chimeric antibodies,
humanized antibodies, Fab, Fab', F(ab')
2, Fv, scFv or dsFv fragments.
[0009] In one aspect of the present invention, the antibody or the antigen-binding fragment
thereof comprises a heavy chain constant region of an amino acid sequence set forth
in SEQ ID NO: 17.
[0010] In one aspect of the present invention, the antibody or the antigen-binding fragment
thereof comprises a heavy chain constant region of an amino acid sequence set forth
in SEQ ID NO: 18.
[0011] In one aspect of the present invention, the antibody or the antigen-binding fragment
thereof comprises a light chain constant region of an amino acid sequence set forth
in SEQ ID NO: 28.
[0012] The antibody or the antigen-binding fragment disclosed herein has one or more of
the following advantages: higher affinity to cells expressing Claudin18.2, enhanced
ability to mediate ADCC, and better tumor inhibitory effect.
[0013] In one aspect of the present invention, any of the above antibodies or the antigen-binding
fragments thereof binds to Claudin18.2.
[0014] The present invention also relates to a chimeric antigen receptor comprising the
antibody or the antigen-binding fragment thereof, a related CAR-T cell, and a preparation
method and use of the same.
[0015] Specifically, in one aspect, the present invention relates to a chimeric antigen
receptor (CAR) comprising any of the above antibodies or the antigen-binding fragments
thereof, wherein three light chain complementarity determining regions of the antibody
or the antigen-binding fragment thereof comprise an LCDR1 amino acid sequence set
forth in SEQ ID NO: 5, an LCDR2 amino acid sequence set forth in SEQ ID NO: 6 and
an LCDR3 amino acid sequence set forth in SEQ ID NO: 7; and three heavy chain complementarity
determining regions of the antibody or the antigen-binding fragment thereof comprise
an HCDR1 amino acid sequence set forth in SEQ ID NO: 8, an HCDR2 amino acid sequence
set forth in SEQ ID NO: 9 and an HCDR3 amino acid sequence set forth in SEQ ID NO:
10.
[0016] In another aspect, the present invention relates to a chimeric antigen receptor (CAR)
comprising an antibody or an antigen-binding fragment thereof, wherein three light
chain complementarity determining regions of the antibody or the antigen-binding fragment
thereof comprise an LCDR1 amino acid sequence set forth in SEQ ID NO: 11, an LCDR2
amino acid sequence set forth in SEQ ID NO: 12 and an LCDR3 amino acid sequence set
forth in SEQ ID NO: 13; and three heavy chain complementarity determining regions
of the antibody or the antigen-binding fragment thereof comprise an HCDR1 amino acid
sequence set forth in SEQ ID NO: 14, an HCDR2 amino acid sequence set forth in SEQ
ID NO: 15 and an HCDR3 amino acid sequence set forth in SEQ ID NO: 16.
[0017] In another aspect, the present invention relates to a chimeric antigen receptor,
wherein the sequence of the VL of the antibody or the antigen-binding fragment thereof
is SEQ ID NO: 1, and the sequence of the VH is SEQ ID NO: 2.
[0018] In another aspect, the present invention relates to a chimeric antigen receptor,
wherein the sequence of the VL of the antibody or the antigen-binding fragment thereof
is SEQ ID NO: 3, and the sequence of the VH is SEQ ID NO: 4.
[0019] In another aspect, the VH and the VL of the antibody or the antigen-binding fragment
thereof are linked through a linker; preferably, through a GGGGSGGGGSGGGGS linker;
preferably, in the order of VH-GGGGSGGGGSGGGGS-VL from N terminus to C terminus.
[0020] In another aspect, the present invention relates to a chimeric antigen receptor which
sequentially comprises the antibody or the antigen-binding fragment thereof according
to any of the preceding aspects, an extracellular hinge region, a transmembrane region
and an intracellular signaling region.
[0021] In another aspect, the present invention relates to a chimeric antigen receptor,
of which the antibody or the antigen-binding fragment thereof is directed by a signal
peptide.
[0022] In another aspect, the present invention relates to a chimeric antigen receptor,
wherein the signal peptide may be a CD8α signal peptide, a VH3 signal peptide, an
IL2 signal peptide or the like, the extracellular hinge region may be a CD8 hinge
region, a CD28 hinge region or the like, the transmembrane region may be a CD8 transmembrane
region, a CD28 transmembrane region, a 4-1BB transmembrane region or the like, and
the intracellular signaling region may be a CD28 signaling region, a 4-1BB signaling
region, an OX40 signaling region, a CD3ζ signaling region or the like.
[0023] In another aspect, the present invention relates to a chimeric antigen receptor,
wherein the extracellular hinge region is a CD8 hinge region, the transmembrane region
is a CD8 transmembrane region, the intracellular signaling region is 4-1BB and CD3ζ,
and the antibody or the antigen-binding fragment thereof is directed by a CD8α signal
peptide. Preferably, the CD8α signal peptide is a CD8α signal peptide set forth in
SEQ ID NO: 21, the extracellular hinge region is a CD8 hinge region set forth in SEQ
ID NO: 22, the transmembrane region is a CD8 transmembrane region set forth in SEQ
ID NO: 23, and the intracellular signaling region is 4-1BB set forth in SEQ ID NO:
24 and CD3ζ set forth in SEQ ID NO: 25.
[0024] In another aspect, the present invention relates to a nucleic acid encoding the antibody
or the antigen-binding fragment thereof, or the chimeric antigen receptor according
to any of the preceding aspects.
[0025] In another aspect, the present invention relates to a vector comprising the nucleic
acid according to the previous aspect, or expressing the antibody or the antigen-binding
fragment thereof or the chimeric antigen receptor according to any of the preceding
aspects. Preferably, the vector may be a viral vector; preferably, the viral vector
includes, but is not limited to, a lentivirus vector, an adenovirus vector, an adeno-associated
virus vector or a retrovirus vector; preferably, the vector may be a non-viral vector;
preferably, the non-viral vector may be a transposon vector; preferably, the transposon
vector may be a Sleeping Beauty vector, a PiggyBac vector, or the like; preferably,
the vector may be a mammalian expression vector; preferably, the expression vector
may be a bacterial expression vector; preferably, the expression vector may be a fungal
expression vector.
[0026] In another aspect, the vector is a lentivirus vector.
[0027] In another aspect, the lentivirus vector is plasmid pRRLSIN-Claudin18.2CAR-P2A-EGFRt
shown in FIG. 11.
[0028] In another aspect, the vector is a PiggyBac (PB) transposon vector.
[0029] In another aspect, the PB transposon vector is plasmid PB CN02 CAR shown in FIG.
24.
[0030] In another aspect, the present invention relates to a cell expressing the antibody
or the antigen-binding fragment thereof or the chimeric antigen receptor according
to any of the preceding aspects. Preferably, the cell is a bacterial cell; preferably,
the bacterial cell is an
Escherichia coli cell or the like; preferably, the cell is a fungal cell; preferably, the fungal cell
is a yeast cell; preferably, the yeast cell is a
Pichia pastoris cell or the like; preferably, the cell is a mammalian cell; and preferably, the mammalian
cell is a Chinese hamster ovary (CHO) cell, a human embryonic kidney cell (293), a
B cell, a T cell, a DC cell, a NK cell, or the like.
[0031] In another aspect, the present invention relates to a CAR-T cell comprising the chimeric
antigen receptor according to any of the preceding aspects.
[0032] In another aspect, the present invention relates to a method for preparing the CAR-T
cell according to the previous aspect, comprising transfecting a T cell with a vector
comprising a nucleic acid encoding the chimeric antigen receptor according to any
of the preceding aspects. In a preferred embodiment, the vector is a non-viral vector.
In a preferred embodiment, the vector is a PB transposon vector. In a preferred embodiment,
the PB transposon vector is plasmid PB CN02 CAR shown in FIG. 24.
[0033] In another aspect, the present invention relates to a method for preparing the CAR-T
cell according to the previous aspect, comprising transfecting a T cell with a vector
comprising a nucleic acid encoding a transposase. In another preferred embodiment,
the transposase is PB transposase.
[0034] In another aspect, the present invention relates to a method for preparing the CAR-T
cell according to the previous aspect, comprising transfecting a T cell with a transposon
vector comprising a nucleic acid encoding the chimeric antigen receptor according
to any of the preceding aspects and a transposase vector comprising a nucleic acid
encoding a transposase. In a preferred embodiment, the transposon vector is a PB transposon
vector. In a preferred embodiment, the PB transposon vector is plasmid PB CN02 CAR
shown in FIG. 24. In a preferred embodiment, the transposase is PB transposase.
[0035] In another aspect, the present invention relates to a method for preparing the CAR-T
cell according to the previous aspect, comprising transducing a T cell with a lentivirus
comprising the chimeric antigen receptor vector according to any of the preceding
aspects to give the CAR-T cell.
[0036] In another aspect, the present invention relates to a pharmaceutical composition
comprising the CAR-T cell according to any of the preceding aspects.
[0037] In another aspect, the present invention relates to a method for treating cancer,
comprising administering the CAR-T cell according to any of the preceding aspects
to a subject in need.
[0038] In another aspect, the present invention relates to use of the CAR-T cell according
to any of the preceding aspects in treating cancer.
[0039] In another aspect, the present invention relates to use of the CAR-T cell according
to any of the preceding aspects in preparing a pharmaceutical composition for treating
cancer.
[0040] In another aspect, the present invention relates to a CAR-T cell having one or more
of the following advantages: good killing ability to cells expressing Claudin18.2;
and low killing ability to cells expressing Claudin18.1.
[0041] In one aspect, the present invention provides a pharmaceutical composition comprising:
the antibody or the antigen-binding fragment thereof, the chimeric antigen receptor,
the nucleic acid encoding the same, or the cell expressing the same disclosed herein;
and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier
includes one or more of the following: pharmaceutically acceptable vehicle, disperser,
additive, plasticizer, and excipient.
[0042] In one aspect, the present invention provides a kit comprising the antibody or the
antigen-binding fragment thereof, a chimeric antigen receptor, or the nucleic acid
encoding the same disclosed herein.
[0043] In some embodiments, the pharmaceutical composition may also comprise other therapeutic
agents. In some embodiments, other therapeutic agents include chemotherapeutic agents,
immunotherapeutic agents, or hormone therapeutic agents. The antibody or the antigen-binding
fragment can be used in combination with other therapeutic agents to enhance the efficacy.
[0044] In some embodiments, "to enhance the efficacy" refers to enhancing the efficacy of
other therapeutic agents or modalities. The antibody or the antigen-binding fragment
disclosed herein can be administered alone or in combination with other therapeutic
agents or modalities. In some embodiments, other therapeutic agents or modalities
include chemotherapeutic agents, immunotherapeutic agents, hormone therapeutic agents,
radiotherapy and surgery.
[0045] In another aspect, the present invention relates to use of the antibody or the antigen-binding
fragment thereof, the chimeric antigen receptor, the nucleic acid, the vector or the
cell according to any of the preceding aspects in preparing a pharmaceutical composition
for treating or preventing a disease.
[0046] In another aspect, the present invention relates to use of the antibody or the antigen-binding
fragment thereof, the chimeric antigen receptor, or the nucleic acid according to
any of the preceding aspects in preparing a kit for diagnosis or detection.
[0047] In another aspect, a method for treating or preventing a disease is provided, comprising
administering the antibody or the antigen-binding fragment, the chimeric antigen receptor,
the nucleic acid, the vector, the cell, or the pharmaceutical composition disclosed
herein to a subject in need.
[0048] In another aspect, a method for diagnosis or detection is provided, comprising administering
the antibody or the antigen-binding fragment, the chimeric antigen receptor, the nucleic
acid, or the kit disclosed herein to a subject in need or a sample.
[0049] In another aspect, the present invention provides use of the antibody or the antigen-binding
fragment thereof, the chimeric antigen receptor, the nucleic acid, the vector, the
cell, or the pharmaceutical composition according to any of the preceding aspects
for treating or preventing a disease.
[0050] In another aspect, the present invention provides use of the antibody or the antigen-binding
fragment thereof, the chimeric antigen receptor, the nucleic acid, or the kit according
to any of the preceding aspects for detection or diagnosis.
[0051] In another aspect, the disease is a cancer.
[0052] In another aspect, the cancer is a Claudin18.2-positive cancer.
[0053] In another aspect, the cancer includes gastric cancer, pancreatic cancer, esophageal
cancer, lung cancer, ovarian cancer, head and neck cancer, bladder cancer, cervical
cancer, sarcoma, cytoma, colon cancer, kidney cancer, colorectal cancer, liver cancer,
melanoma, breast cancer, myeloma, neuroglioma, leukemia, lymphoma, and the like.
DRAWINGS
[0054]
FIGs. 1-2 show the binding sensitivity of candidate antibodies to Claudin18.2 protein
measured by ELISA.
FIGs. 3A-3B show the binding of candidate antibodies to 293T-Claudin18.2 cells measured
by flow cytometry.
FIGs. 4A-4B show the binding of candidate antibodies to 293T-Claudin18.1 cells measured
by flow cytometry.
FIGs. 5A-5B show the binding of candidate antibodies to NUGC4 cells measured by flow
cytometry.
FIGs. 6A-6B show the ADCC of candidate antibodies on 293T-Claudin18.2 cells.
FIGs. 7A-7B show the ADCC of candidate antibodies on NUGC4 cells.
FIG. 8 shows the ADCC of candidate antibodies on 293T-Claudin18.1 cells.
FIG. 9 shows the pharmacodynamics of candidate antibodies.
FIG. 10 shows a structural schematic of plasmid pRRLSIN-EGFRt.
FIG. 11 shows a structural schematic of recombinant plasmid pRRLSIN-Claudin18.2CARP2A-EGFRt.
FIG. 12 shows the lentivirus activity titers of different chimeric antigen receptors.
FIG. 13 shows the positive rates of T lymphocytes expressing different chimeric antigen
receptors.
FIG. 14 shows the specific lysis assay of Claudin18.2 CAR-T cells with different scFvs
in vitro.
FIG. 15 shows the release of IFN-gamma cytokines in supernatant of co-incubated Claudin18.2
CAR-T cells with different scFvs and different 293T cells.
FIG. 16 shows the specific lysis assay of Claudin18.2 CAR-T cells with different scFvs
against Claudin18.2-positive tumor cells.
FIG. 17 shows the release of IFN-gamma cytokines in supernatant of co-incubated Claudin18.2
CAR-T cells with different scFvs and Claudin18.2-positive tumor cells.
FIG. 18 shows the tumor volumes in female NOG mice bearing NUGC4-Claudin18.2 tumor
cells after receiving Claudin18.2 CAR-T cells.
FIG. 19 shows the body weight of female NOG mice bearing NUGC4-Claudin18.2 tumor cells
after receiving Claudin18.2 CAR-T cells.
FIG. 20 shows the tumor growth inhibition (TGI) in female NOG mice bearing NUGC4-Claudin18.2
tumor cells after receiving Claudin18.2 CAR-T cells.
FIG. 21 shows the survival rate of female NOG mice bearing NUGC4-Claudin18.2 tumor
cells after receiving Claudin18.2 CAR-T cells.
FIG. 22 shows the positive rates of T lymphocytes expressing different chimeric antigen
receptors.
FIG. 23 shows the in vitro specific lysis assay of CN02 CAR-T cells prepared by different methods.
FIG. 24 shows the structural schematic of plasmid PB CN02 CAR.
DETAILED DESCRIPTION
[0055] The present invention will be further described in conjunction with the following
specific examples. The examples described herein are only some examples of the present
invention, but not all examples. It should be understood that the following examples
are given to provide those of ordinary skill in the art a complete disclosure and
description of how to utilize the methods and the compositions, but are not intended
to limit the scope of the present invention. Based on the examples of the present
invention, all other examples obtained by those of ordinary skill in the art without
creative work shall fall within the protection scope of the present invention.
Example 1. Production of Anti-Claudin18.2 Monoclonal Antibody
1.1 Immunization
[0056] BoAn-hMab transgenic mice from Shandong BoAn Biotechnology Co. Ltd. (prepared according
to the method described in Chinese Patent No.
CN103571872B) were immunized with plasmids (KYinno) containing Claudin18.2 genes and CHO cells
(KYinno) stably expressing Claudin18.2 protein. Plasmids were used for the first immunization,
and the second to the seventh immunizations were conducted using plasmids and cells
alternately. A total of 10 mice were immunized. Five mice with higher serum concentration
were selected for booster immunization, and the mice were euthanized 4 days later.
Spleens were processed and frozen for later use.
1.2 Construction of phage library
[0057] Spleen cells of the immunized mice were added with Trizol (Thermo Scientific, catalog
number: 15596-026) for complete lysis, and then added with a 1/5 volume of chloroform
and mixed well. The mixture was incubated at room temperature for 20 min, and centrifuged
at 12000 rpm at 4 °C for 20 min. The supernatant was added with an equal volume of
isopropanol. The resulting mixture was incubated at room temperature for 20 min and
then centrifuged at 12000 rpm at 4 °C for 20 min. The supernatant was discarded, and
the precipitate was washed with 75% ethanol twice and then centrifuged at 12000 rpm
at 4 °C for 5 min. The supernatant was discarded, and the precipitate was dried at
room temperature and then resuspended with DEPC water to give RNA, which was then
reversely transcribed into cDNA using a Roche reverse transcription kit Transcriptor
First Strand cDNA Synthesis Kit as per the instructions (Roche Applied Science, catalog
number: 4897030001).
[0058] The phage library was constructed by the method described in Carlos F. Barbas III,
Phage display: A Laboratory Manual. Sequences of variable regions of heavy and light chains were obtained from cDNA by
PCR, and then subjected to overlap extension PCR to give scFv sequence. Then, the
scFv was digested with SfiI enzyme (NEB, catalog number: R0123L) for 5 h (50 °C) and
ligated with plasmid pCOMB3x (Biovector Science Lab, Inc., BIOVECTOR510837) through
T4 DNA ligase (Sino Biological Inc.). The ligation product was electrotransfected
into competent
Escherichia coli TG1 cells (Lucigen, catalog number: A96595-2), which were then cultivated on a shaker
at 220 rpm at 37 °C and infected with a phage, and the supernatant of the culture
was collected, concentrated and purified to give the phage library.
1.3 Screening
[0059] 1.3.1 Plate screening: A plate was coated with Claudin18.2 protein (Genscript Biotech)
at 0.3 µg/well, and incubated overnight at 4 °C. The plate was blocked with 2% BSA
for 1 h the next day, and the phage library (2 × 10
12) was added for a 2-h incubation. After 4-10 washings, the phages specifically binding
to Claudin18.2 were eluted with an eluent buffer (pH 2.2) (4.2 mL of concentrated
hydrochloric acid (Tianjin Kemiou Chemical Reagent Co., Ltd.) was added to 500 mL
of ultrapure water, and the mixture was adjusted to pH 2.2 with glycine powder (Biotopped,
BG0617-500)).
[0060] 1.3.2 Cell screening: The phage library (2 × 10
12) was rotationally mixed with 293T-Claudin18.1 cells (3 × 10
6 cells/vial) at room temperature and incubated for 1 h. The resulting mixture was
blocked with 2% BSA for 1 h, then rotationally mixed with 293T-Claudin18.2 cells (2
× 10
6 cells/vial) at room temperature and incubated for 2 h. After 4-10 washings, the phages
specifically binding to Claudin18.2 were eluted with an eluent buffer (pH 2.2). Phages
passing the cell screening could be further screened on plates.
Example 2. Construction and production of complete antibodies
[0061] Clones CLD387-C115, CLD389-C279\CA802\CA852, CLDQMix-CA808.1\CA811\CA818, CLDQ1-CA841\CA843,
CLD393-C1002\C1024 were sequenced by Invitrogen Biotechnology Co., Ltd. The amino
acid sequences of the variable regions of the clones are shown in Table 1.
[0062] Through methods such as variable region gene amplification (2
∗Phanta Max Master Mix, manufacturer: Vazyme, catalog number: P515-AA, batch number:
7E211GB), signal peptide and variable region overlap extension, and homologous recombination
(ClonExpress II One Step Cloning Kit, manufacturer: Vazyme, catalog number: C112-01,
batch number: 7E211L8), nucleotide sequence fragments encoding VH or VL were respectively
inserted into vectors pCDNA3.4 (Life Technology) containing a nucleotide sequence
encoding a heavy chain constant region (SEQ ID NO: 17) and pCDNA3.4 (Life Technology)
containing a nucleotide sequence encoding a light chain constant region (SEQ ID NO:
28). Then the vectors were transfected into HEK293 cells and incubated on a shaker
at 37 °C/8% CO
2/125 rpm. After 6-7 days of transient expression the supernatant was purified by Protein
A affinity chromatography to give Claudin18.2 antibodies, and the antibody concentration
was determined by the extinction coefficient at UV280.
[0063] Antibody IMAB362 was selected as the reference antibody. In the Phase II clinical
trial for gastric cancer, the antibody in combination with chemotherapy significantly
prolonged the survival (13.2 vs. 8.4 months) as compared with standard chemotherapy.
IMAB362 had more significant efficacy and a longer median survival time (16.7 months)
in patients with high expression of Claudin18.2. IMAB362 is also one of the first
Claudin18.2 antibodies put into clinical trials.
[0064] Production of reference antibody: Amino acid sequence of Claudin18.2 antibody IMAB362
of Ganymed is available in IMGT database and the Patent No.
CN201380026898, with the heavy chain and light chain sequences set forth in SEQ ID NOs: 19 and 20,
respectively. The whole gene sequence was synthesized, inserted in vector pCDNA3.4
and expressed in HEK293 cells to produce an antibody named IMAB362.
Example 3. Characterization of Anti-Claudin18.2 Antibodies
3.1 Binding of antibodies to Claudin18.2 protein by Elisa
[0065] Plates were coated with Claudin18.2 antigen (Genscript Biotech) of different concentrations
(0.2 µg/mL, 0.05 µg/mL, 0.0125 µg/mL) at 100 µL/well, incubated overnight at 4 °C,
and blocked with 3% skimmed milk powder at 37 °C for 1 h. 100 µL of candidate antibodies
was added to each well at 1 µg/mL, and incubated at 37 °C for 1 h, followed by goat
anti-human IgG/HRP and a 1-h incubation at 37 °C. After a 10-min color development,
OD
450 was measured on a microplate reader. The results are shown in FIG. 1, FIG. 2, Table
2 and Table 3.
Table 2. Binding sensitivity of candidate antibodies to Claudin18.2 protein measured
by ELSIA
Antibody ID |
Antigen concentration (0.2 µg/mL) |
Antigen concentration (0.05 µg/mL) |
Antigen concentration (0.0125 µg/mL) |
CLDQ1-CA843-IgG1 |
1.847 |
0.452 |
0.148 |
CLD387-115-IgG1 |
2.724 |
1.66 |
0.449 |
CLD389-279-IgG1 |
3.094 |
2.15 |
0.702 |
CLD389-CA802-IgG1 |
2.618 |
1.622 |
0.579 |
CLDMIX-CA808.1-IgG1 |
2.996 |
1.167 |
0.389 |
CLDMIX-CA811-IgG1 |
2.705 |
1.062 |
0.307 |
CLDQ1-CA841-IgG1 |
2.38 |
0.484 |
0.165 |
CLD389-CA852-IgG1 |
2.809 |
1.17 |
0.332 |
IMAB362 |
2.819 |
1.475 |
0.532 |
Table 3. Binding sensitivity of candidate antibodies to Claudin18.2 protein measured
by ELSIA
Antibody ID |
Antigen concentration (0.2 µg/mL) |
Antigen concentration (0.05 µg/mL) |
Antigen concentration (0.0125 µg/mL) |
CLD383-C1024-IgG1 |
0.212 |
0.109 |
0.084 |
CLD387-C1002-IgG1 |
0.187 |
0.105 |
0.076 |
CLDMix-CA818-IgG1 |
3.189 |
1.281 |
0.492 |
IMAB362 |
3.832 |
3.288 |
1.901 |
3.2 Binding of antibodies to 293T-Claudin18.1/18.2 cells and NUGC4 cells measured
by flow cytometry
[0066] To a 96-well round-bottom plate, 50 µL of 293T-Claudin18.1 or 18.2 cells (KYinno)
or NUGC4 cells were added at 1 × 10
5 cells/well. Each candidate antibody was serially diluted with FACS buffer (sterile
PBS, 0.2% BSA), and added to the 96-well round-bottom plate at 50 µL/ well before
an incubation at 4 °C for 1 h. The supernatant was discarded after centrifugation
at 2000 rpm for 3 min. Then the resulting cells were washed twice with FACS buffer,
added with 100 µL/well of fluorescent secondary antibody (Southern Biotech, 2040-09),
with a final concentration of 1 µg/mL, and incubated at 4 °C for 1 h before a centrifugation
at 2000 rpm for 3 min. The supernatant was discarded, and the resulting cells were
washed twice with FACS buffer, resuspended with 100 µL/well of FACS buffer, and analyzed
by a flow cytometer (ACEA Pharma, NovoCyte 2060). The results are shown in FIGs. 3A-5B
and Tables 4-9.
[0067] As shown in FIG. 3A, with 293T-Claudin18.2 cells being the target, the mean fluorescence
intensity of candidate antibody CLDQMIX-CA808.1-IgG1 was higher than that of IMAB362
at concentrations of 3 µg/mL, 1.5 µg/mL, 0.75 µg/mL and 0.375 µg/mL. This indicates
that the candidate antibody CLDQMIX-CA808.1-IgG1 has a higher affinity to 293T-Claudin18.2
cells at concentrations of 3 µg/mL, 1.5 µg/mL, 0.75 µg/mL and 0.375 µg/mL.
[0068] As shown in FIG. 5B, with NUGC4 cells being the target, the mean fluorescence intensity
of candidate antibody CLDQMIX-CA808.1-IgG1 was higher than that of IMAB362 at concentrations
of 3 µg/mL, 1.5 µg/mL, and 0.75 µg/mL. This indicates that the candidate antibody
CLDQMIX-CA808.1-IgG1 has a higher affinity to NUGC4 cells expressing Claudin18.2 at
concentrations of 3 µg/mL, 1.5 µg/mL, and 0.75 µg/mL.
[0069] As shown in FIG. 3A, with 293T-Claudin18.2 cells being the target, the mean fluorescence
intensity of the candidate antibody CLDQ1-CA841-IgG1 was higher than that of IMAB362
at concentrations of 3 µg/mL and 1.5 µg/mL. This indicates that the candidate antibody
CLDQ1-CA841-IgG1 has a higher affinity to 293T-Claudin18.2 cells at concentrations
of 3 µg/mL and 1.5 µg/mL.
[0070] As shown in FIG. 4A, with 293T-Claudin18.1 cells being the target, the mean fluorescence
intensities of the candidate antibodies CLDQMIX-CA808.1-IgG1 and CLDQ1-CA841-IgG1
were similar to that of IMAB362, which were all at lower levels at various concentrations.
[0071] The above results show that CLDQMIX-CA808.1-IgG1 and CLDQ1-CA841-IgG1 have better
ability of binding cells expressing Claudin18.2, and have weak ability of binding
Claudin18.1. It indicates that Claudin18.2 cells are more prone to binding, and specific
binding to targets other than Claudin18.2 is less likely to occur in clinical applications,
thereby achieving better pharmaceutical effects.
Table 4. Binding of candidate antibodies to 293T-Claudin18.2 cells measured by flow
cytometry (corresponding to FIG. 3A)
Antibody ID |
Antibody concentration (3 µg/mL) |
Antibody concentration (1.5 µg/mL) |
Antibody concentration (0.75 µg/mL) |
Antibody concentration (0.375 µg/mL) |
CLD387-115-IgG1 |
952384 |
1002923 |
714249 |
501750 |
CLD389-279-IgG1 |
1239649 |
1057051 |
775956 |
487518 |
CLD389-CA802-IgG1 |
1226148 |
1049205 |
773824 |
494669 |
CLDQMIX-CA808.1-IgG1 |
1382494 |
1196101 |
752733 |
535348 |
CLDMIX-CA811-IgG1 |
806980 |
657103 |
504375 |
324014 |
CLDQ1-CA841-IgG1 |
1092233 |
854886 |
583477 |
307641 |
CLDQ1-CA843-IgG1 |
609424 |
603959 |
399820 |
182880 |
CLD389-CA852-IgG1 |
741760 |
577688 |
364883 |
196431 |
IMAB362 |
902134 |
849017 |
688504 |
497940 |
Table 5. Binding of candidate antibodies to 293T-Claudin18.2 cells measured by flow
cytometry (corresponding to FIG. 3B)
Antibody ID |
Antibody concentration (3 µg/mL) |
Antibody concentration (1.5 µg/mL) |
Antibody concentration (0.75 µg/mL) |
Antibody concentration (0.375 µg/mL) |
CLDMix-CA818-IgG1 |
33274 |
34210 |
17288 |
9356 |
CLD393-C1002-IgG1 |
1326330 |
1156050 |
852105 |
405515 |
CLD393-C1024-IgG1 |
1374048 |
1030045 |
787017 |
308011 |
IMAB362 |
1037843 |
877912 |
649900 |
407382 |
Table 6. Binding of candidate antibodies to 293T-Claudin18.1 cells measured by flow
cytometry (corresponding to FIG. 4A)
Antibody ID |
Antibody concentration (3 µg/mL) |
Antibody concentration (1.5 µg/mL) |
Antibody concentration (0.75 µg/mL) |
Antibody concentration (0.375 µg/mL) |
CLD387-115-IgG1 |
124974 |
133585 |
78691 |
63446 |
CLD389-279-IgG1 |
128702 |
72429 |
52042 |
36901 |
CLD389-CA802-IgG1 |
193720 |
162744 |
140589 |
96678 |
CLDQMIX-CA808.1-IgG1 |
47521 |
28885 |
29387 |
26004 |
CLDMIX-CA811-IgG1 |
114724 |
93551 |
40496 |
26022 |
CLDQ1-CA841-IgG1 |
10958 |
37855 |
18119 |
21489 |
CLDQ1-CA843-IgG1 |
86333 |
59051 |
35542 |
23218 |
CLD389-CA852-IgG1 |
70114 |
72110 |
40071 |
18876 |
IMAB362 |
35584 |
24896 |
31961 |
15638 |
Table 7. Binding of candidate antibodies to 293T-Claudin18.1 cells measured by flow
cytometry (corresponding to FIG. 4B)
Antibody ID |
Antibody concentration (3 µg/mL) |
Antibody concentration (1.5 µg/mL) |
Antibody concentration (0.75 µg/mL) |
Antibody concentration (0.375 µg/mL) |
CLDMix-CA818-IgG1 |
34418 |
17935 |
15184 |
7957 |
CLD393-C1002-IgG1 |
15767 |
8615 |
12343 |
6963 |
CLD393-C1024-IgG1 |
10099 |
17142 |
9851 |
7892 |
IMAB362 |
12483 |
17319 |
20666 |
12817 |
Table 8. Binding of candidate antibodies to NUGC4 cells measured by flow cytometry
(corresponding to FIG. 5A)
Antibody ID |
Antibody concentration (3 µg/mL) |
Antibody concentration (1.5 µg/mL) |
Antibody concentration (0.75 µg/mL) |
CLD387-115-IgG1 |
201377 |
126584 |
101395 |
CLD389-279-IgG1 |
158296 |
78264 |
40032 |
CLD389-CA802-IgG1 |
113085 |
58148 |
24412 |
CLDMIX-CA811-IgG1 |
43084 |
26853 |
15543 |
CLDMix-CA818-IgG1 |
42617 |
20432 |
15029 |
CLDQ1-CA841-IgG1 |
32421 |
13208 |
11119 |
IMAB362 |
130923 |
97087 |
69564 |
Table 9. Binding of candidate antibodies to NUGC4 cells measured by flow cytometry
(corresponding to FIG. 5B)
Antibody ID |
Antibody concentration (3 µg/mL) |
Antibody concentration (1.5 µg/mL) |
Antibody concentration (0.75 µg/mL) |
CLDQMIX-CA808.1-IgG1 |
185377 |
137723 |
75649 |
CLDQ1-CA843-IgG1 |
13935 |
10612 |
7234 |
CLD389-CA852-IgG1 |
18351 |
11955 |
9229 |
CLD393-C1002-IgG1 |
5170 |
4891 |
4946 |
CLD393-C1024-IgG1 |
6141 |
5643 |
5210 |
IMAB362 |
116862 |
93509 |
55403 |
3.3 ADCC of antibodies
[0072] Sterile fetal bovine serum was thawed and added to an RPMI1640 medium at a ratio
of 1:99 to give an ADCC buffer. PBMC cells were thawed and incubated overnight in
an incubator at 37 °C/5% CO
2. The density of target cells (293T-Claudin18.1 or 18.2) was adjusted with the ADCC
Buffer to 2 × 10
5 cells/mL, and 50 µL of the target cells was added to each well of a 96-well round-bottom
plate. The antibodies to be tested were diluted by 10× with the ADCC Buffer from 10
µg/mL or 50 µg/mL, then 50 µL of the diluted antibody was added to each well of the
96-well round-bottom plate coated with the target cells, and incubated in an incubator
at 37 °C/5% CO
2 for 30-60 min. PBMC cells were collected and diluted with the ADCC buffer to a density
from 2 × 10
6 cells/mL to 5 × 10
6 cells/mL, then 100 µL of the diluted cells was added to each well of the 96-well
round-bottom plate coated with the target cells and the sample to be tested, and incubated
in an incubator at 37 °C/5% CO
2 for 4-6 h. After incubation, the cells were centrifuged at 300 g for 2-5 min, then
50 µL of supernatant was carefully pipetted to a new 96-well flat-bottom plate, and
50 µL of LDH test solution (Promega, G1780) was added. The cells were then incubated
in an incubator at 37 °C/5% CO
2 for 30 min. A terminating buffer was added after incubation. The OD value at 490
nm was measured by a microplate reader, with a background wavelength being 650 nm.
The results are shown in FIGs. 6A-6B and Tables 10-11.
[0073] As shown in FIG. 6A, with 293T-Claudin18.2 as target and PBMC as effector cell, the
inhibition rate of the candidate antibody CLDQMIX-CA808.1-IgG1 to the target cells
at concentrations of 1 µg/mL and 0.1 µg/mL was higher than that of the reference antibody
IMAB362 at the corresponding concentrations, suggesting that the candidate antibody
CLDQMIX-CA808.1-IgG1 has a better ability of mediating ADCC than the reference antibody
IMAB362 at the concentrations of 1 µg/mL and 0.1 µg/mL. This indicates that CLDQMIX-CA808.1-IgG1
can kill target cells expressing Claudin18.2 better and has better pharmaceutical
effects.
Table 10. ADCC of candidate antibodies on 293T-Claudin18.2 cells (corresponding to
FIG. 6A)
|
Inhibition rate, % |
Antibody ID |
Antibody concentration (10 µg/mL) |
Antibody concentration (1 µg/mL) |
Antibody concentration (0.1 µg/mL) |
CLD387-115-IgG1 |
15.48077 |
21.48077 |
12.28077 |
CLD389-279-IgG1 |
40.95 |
16.05 |
7.530769 |
CLD389-CA802-IgG1 |
61.21154 |
61.38846 |
25.60769 |
CLDQMIX-CA808.1-IgG1 |
57.43462 |
63.55769 |
39.42692 |
CLDMIX-CA811-IgG1 |
63.24208 |
33.91539 |
18.17308 |
CLDQ1-CA841-IgG1 |
58.48615 |
27.23462 |
15.64231 |
CLDQ1-CA843-IgG1 |
53.19346 |
18.85385 |
-2.088462 |
CLD389-CA852-IgG1 |
39.92692 |
12.28077 |
-0.2115385 |
IMAB362 |
60.56292 |
41.36427 |
20.82923 |
Table 11. ADCC of candidate antibodies on 293T-Claudin18.2 cells (corresponding to
FIG. 6B)
|
Inhibition rate, % |
Antibody ID |
Antibody concentration (50 µg/mL) |
Antibody concentration (5 µg/mL) |
Antibody concentration (0.5 µg/mL) |
CLDMix-CA818-IgG1 |
45 |
25 |
15 |
CLD393-C1002-IgG1 |
64 |
49 |
22 |
CLD393-C1024-IgG1 |
48 |
47 |
15 |
IMAB362 |
49 |
45 |
18 |
[0074] Cells were counted and diluted with an ADCC buffer to 4 × 10
5 cells/mL. A proper amount of the sample was taken for a serial dilution. Effector
cells Jurkat (G7011, Promega) were centrifuged at 1500 rpm with the supernatant discarded,
and resuspended with 1% FBS RPMI-1640 medium. The cells were counted and diluted with
the ADCC buffer to 8 × 10
5 cells/mL. Then 25 µL of the target cells was added to each well of a white 96-well
plate (3917, Costar), and 25 µL of the serially diluted antibodies was added to each
well coated with the target cells. 25 µL of effector cells (Jurkat) was added to each
well in a ratio of effector cells to target cells of 20000:10000. Then the 96-well
plate was incubated in a cell incubator for 5 h, and equilibrated at room temperature.
Then 75 µL of a Bio-Glo color-developing buffer (G7940, Promega) was added to each
well for a 15-min reaction, and the plate was detected on a Tecan microplate reader
(chemiluminescence). The results are shown in FIGs. 7A-8 and Tables 12-13.
[0075] As shown in FIG. 7A, with NUGC4 being the target, the EC
50 value of CLDQMIX-CA808.1-IgG1 was 1.264 µg/mL, which was less than that of the reference
antibody IMAB362 of 2.154 µg/mL, indicating that the candidate antibody CLDQMIX-CA808.1-IgG1
has a better ability of mediating ADCC than the reference antibody IMAB362. This indicates
that CLDQMIX-CA808.1-IgG1 can kill target cells expressing Claudin18.2 better and
has better pharmaceutical effects.
Table 12. ADCC of candidate antibodies on NUGC4 cells (corresponding to FIG. 7A)
Antibody ID |
EC50 (µg/mL) |
Antibody ID |
EC50 (µg/mL) |
CLDQMIX-CA808.1-IgG1 |
1.264 |
CLD387-115-IgG1 |
14.27 |
CLDQ1-CA841-IgG1 |
17.33 |
IMAB362 |
2.154 |
Table 13. ADCC of candidate antibodies on NUGC4 cells (corresponding to FIG. 7B)
Antibody ID |
EC50 (µg/mL) |
Antibody ID |
EC50 (µg/mL) |
CLD389-279-IgG1 |
0.81 |
IMAB362 |
2.56 |
CLD389-CA802-IgG1 |
15.73 |
/ |
/ |
Example 4. Modification of Fc Terminus of Anti-Claudin 18.2 Antibodies
4.1 Modification of Fc terminus of antibodies
[0076] In order to enhance the ADCC of antibodies and change the affinity between the antibodies
and Fc receptor, the Fc terminus of the antibody was mutated. The mutated amino acid
sequence of heavy chain constant region is set forth in SEQ ID NO: 18. The resulting
antibody was named CLDQMix-CA808.1-IgG1-VLPLL.
4.2 Affinity of modified antibody to Fc receptors
4.2.1 Affinity of antibody to human FcRn
[0077] The antibody binding kinetics was determined by Octet
RED 96 system based on the biolayer interferometry (BLI). Human FcRn (ACROBiosystems,
FCM-H82W4, 1 µg/mL) was loaded to Streptavidin (SA) Dip and Read
™ Biosensors with a loading height of 0.2 nm. The antibody was serially diluted by
2× with PBST from 33.3 mM, and a blank control was set. The Association time was set
to 150 s, and the dissociation time was set to 100 s. After the assay, the equilibrium
dissociation constant (k
D) was calculated using Steady State Analysis.
4.2.2 Affinity of antibody to human CD32a(H)
[0078] The antibody binding kinetics was determined by Octet
RED 96 system based on the biolayer interferometry (BLI). Human CD32a(H) (Sino Biological,
10374-H27H1-B, 1 µg/mL) was loaded to Streptavidin (SA) Dip and Read
™ Biosensors with a loading height of 0.2 nm. The antibody was serially diluted by
2× with PBST from 1000 mM, and a blank control was set. The Association time was set
to 150 s, and the dissociation time was set to 100 s. After the assay, the equilibrium
dissociation constant (k
D) was calculated using Steady State Analysis.
4.2.3 Affinity of antibody to human CD16a(V)
[0079] The antibody binding kinetics was determined by Octet
RED 96 system based on the biolayer interferometry (BLI). Human CD16a(V) (Sino Biological,
10389-H27H1-B, 0.5 µg/mL) was loaded to Streptavidin (SA) Dip and Read
™ Biosensors with a loading height of 0.5 nm. The antibody was serially diluted by
2× with PBST from 166.7 mM, and a blank control was set. The Association time was
set to 30 s, and the dissociation time was set to 100 s. After the assay, association
constant (k
on), dissociation constant (k
dis) were calculated by curve fitting with a 1:1 model and equilibrium dissociation constant
(k
D) were calculated in the ratio of k
d/k
a.
4.2.4 Affinity of antibody to human CD16a(F)
[0080] The antibody binding kinetics was determined by Octet
RED 96 system based on the biolayer interferometry (BLI). Human CD16a(F) (Sino Biological,
10389-H27H-B, 0.5 µg/mL) was loaded to Streptavidin (SA) Dip and Read
™ Biosensors with a loading height of 0.5 nm. The antibody was serially diluted by
2× with PBST from 333.3 mM, and a blank control was set. The Association time was
set to 30 s, and the dissociation time was set to 100 s. After the assay, association
constant (k
on), dissociation constant (k
dis) were calculated by curve fitting with a 1:1 model and equilibrium dissociation constant
(k
D) were calculated in the ratio of k
d/k
a.
4.2.5 Affinity of antibody to human CD32b
[0081] The antibody binding kinetics was determined by Octet
RED 96 system based on the biolayer interferometry (BLI). Human CD32a(H) (Sino Biological,
10374-H27H1-B, 1 µg/mL) was loaded to Streptavidin (SA) Dip and Read
™ Biosensors with a loading height of 0.2 nm. The antibody was serially diluted by
2× with PBST from 2000 mM, and a blank control was set. The Association time was set
to 40 s, and the dissociation time was set to 50 s. After the assay, the equilibrium
dissociation constant (k
D) was calculated using Steady State Analysis.
[0082] As shown in Table 14, the affinity of CLDQMIX-CA808.1-IgG1-VLPLL to agonistic receptors,
particularly Human CD16a(F), was greatly improved, thus better promoting the ADCC
in subjects.
Table 14. Affinity of candidate antibody to Fc receptors
|
FC receptor |
KD (M) of CLDQMIX-CA808.1-IgG1 |
KD (M) of CLDQMIX-CA808.1-IgG1-VLPLL |
Ratio of KD |
Agonistic receptors |
Human CD32a(H) |
4.10E-07 |
3.00E-07 |
1.37↑ |
Human CD16a(V) |
1.07E-07 |
5.41E-08 |
1.98↑ |
Human CD16a(F) |
5.66E-07 |
1.19E-07 |
4.75↑ |
Human FcRn |
6.93E-09 |
5.27E-09 |
1.31↑ |
Inhibitory receptor |
Human CD32b |
3.20E-06 |
3.00E-06 |
- |
Example 5. Pharmacodynamics of Anti-Claudin18.2 Antibodies
[0083] Human gastric cancer NUGC4 cells (JCRB Cell Bank, catalog number: JCRB0834) were
cultivated in an RPMI1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin
and 100 µg/mL streptomycin through monolayer culture
in vitro in an incubator at 37 °C/5% CO
2. The cells were digested with trypsin-EDTA twice a week for passaging as per conventional
practice. At a cell saturation of 80%-90% and a required number, the cells were harvested,
counted and grafted into BALB/c nude mice (female, 6-8 weeks old, 18-22g) (Shanghai
Lingchang Biotechnology Co., Ltd.). 0.2 mL (1 × 10
6 cells) of NUGC4 cells (along with matrigel in a volume ratio of 1:1) was subcutaneously
grafted on the right back of each mouse, and the mice were randomized when the mean
tumor volume was approximately 60-70 mm
3. The animals were weighed before administration and the tumor volume was measured.
The mice were randomized by the tumor volume (randomized block design), 8 in each
group. The weight was measured twice a week, and the tumor diameter was measured with
a vernier caliper twice a week. The tumor volume was calculated using the following
formula: V = 0.5a × b
2, where, a and b represent the long diameter and short diameter of the tumor respectively.
The results are shown in FIG. 9 and Table 15.
[0084] As shown in FIG. 9, the tumor inhibitory effect of 5 mg/kg CLDQMIX-CA808.1-IgG1-VLPLL
was comparable to that of 10 mg/kg IMAB362. Compared with 10 mg/kg IMAB362, 10 mg/kg
CLDQMIX-CA808.1-IgG1-VLPLLdemonstrated better tumor inhibitory effect. This indicated
that the CLDQMIX-CA808.1-IgG1-VLPLLantibody has better tumor inhibitory activity than
IMAB362, and the inhibitory effect of CLDQMIX-CA808.1-IgG1- VLPLL on tumors is dose-dependent.
The tumor inhibitory effect increases with the dose.
Table 15. Pharmacodynamics of candidate antibodies (corresponding to FIG. 9)
Antibody ID |
Final tumor volume (mm3) |
Vehicle |
1536.5±195.8 |
IMAB362 10mg/kg |
1301.0±177.2 |
CLDQMIX-CA808.1-IgG1-VLPLL 5mg/kg |
1307.8±186.4 |
CLDQMIX-CA808. 1 -IgG1 -VLPLL 10mg/kg |
1103.3±186.5 |
CLDQMIX-CA808. 1 -IgG1 -VLPLL 20mg/kg |
1006.4±207.1 |
Example 6. Preparation of Claudin18.2-Specific Chimeric Antigen Receptor-Modified
T Cells 6.1 Preparation of gene fragment of chimeric antigen receptor
[0085] In the present invention, a fusion gene fragment was designed in the following order
of coding genes: CD8α signal peptide, CA841 scFv VH-linker-CA841 scFv VL, CD8 hinge
region, CD8 transmembrane region, and 4-1BB and CD3ζ intracellular signaling regions,
and the fusion gene was directly synthesized by gene synthesis techniques, allowing
the expressed chimeric antigen receptor to have an amino acid sequence of scFv VH-linker-scFv
VL-CD8 hinge-CD8TM-4-1BB-CD3ζ. The linker had a sequence of GGGGSGGGGSGGGGS, the CD8α
signal peptide had a sequence of SEQ ID NO: 21, the CD8 hinge region (CD8 hinge) had
a sequence of SEQ ID NO: 22, the CD8 transmembrane region (CD8 TM) had a sequence
of SEQ ID NO: 23, the 4-1BB had a sequence of SEQ ID NO: 24, and the CD3ζ had a sequence
of SEQ ID NO: 25.
[0086] The whole gene of pRRLSIN lentivirus vector containing a human EFla promoter was
synthesized, and the green fluorescent protein (GFP) sequence was replaced by an EGFRt
marker protein sequence to give a pRRLSIN-EGFRt vector (see FIG. 10).
6.2 Construction of lentiviral expression vector of chimeric antigen receptor
[0087] In the example, the vector system used to construct the lentivirus plasmid vector
of the present invention was a third generation self-inactivated lentivirus vector
system. The system has three plasmids: a pMDLg-pRRE packaging plasmid (Unibio, VT1449)
encoding protein Gag/Pol, a pRSV-rev packaging plasmid (Unibio, VT1445) encoding Rev
protein, and an envelope plasmid PMD2.G (Unibio, VT1443) encoding VSV-G protein.
[0088] In the example, a lentivirus expression vector expressing specific CAR and EGFRt
(SEQ ID NO: 27) linked by P2A (SEQ ID NO: 26) was constructed, and the target gene
obtained in Section 6.1 was linked to the pRRLSIN-EGFRt vector to form a recombinant
plasmid named pRRLSIN-Claudin18.2CAR-P2A-EGFRt (see FIG. 11). The specific sequence
was pRRLSIN-CD8α-scFv VH-linker-scFv VL-CD8hinge-CD8TM-4-1BB-CD3ζ-P2A-EGFRt. After
verified by enzyme digestion and sequencing, the successfully constructed vector was
ready to package. CAR-P2A-EGFRt was transcribed into a single mRNA, but finally translated
into two peptide chains of EGFRt and anti-Claudin18.2 chimeric antigen receptor. Anti-Claudin18.2
CAR was located on the cell membrane under the direction of the CD8α signal peptide.
[0089] The four sequences containing the target CAR obtained in the example are as follows:
scFv CA808.1-CD8hinge-CD8TM-4-1BB-CD3ζ-P2A-EGFRt (hereinafter referred to as CN01)
scFv CA841-CD8hinge-CD8TM-4-1BB-CD3ζ-P2A-EGFRt (hereinafter referred to as CN02)
scFv C279-CD8hinge-CD8TM-4-1BB-CD3ζ-P2A-EGFRt (hereinafter referred to as CN03)
scFv C115-CD8hinge-CD8TM-4-1BB-CD3ζ-P2A-EGFRt (hereinafter referred to as CN04)
6.3 Preparation of chimeric antigen receptor lentivirus
[0090] The pRRLSIN-Claudin18.2CAR-P2A-EGFRt expression plasmid and pMDLg-pRRE, pRSV-rev
and pMD2.G helper plasmids were extracted and mixed with the transfection reagent
polyethyleneimine (PEI) in a certain ratio to co-transfect 293T cells. The major procedures
are as follows:
- (1) The 293T cells passaged to 5-8th generations (ATCC CRL-3216) were seeded at a
cell density of 7 × 106 in a DMEM medium (purchased from GIBCO) containing 10% FBS (purchased from GIBCO)
in 75 cm3 cell culture flasks. After mixing, the cells were cultivated in a CO2 incubator at 37 °C/5% CO2 for 24 h before transfection. A cell aggregation of about 70-80% was observed on
the next day, and the cells were transfected.
- (2) 24h later, the target expression plasmid and the pMDLg-pRRE, pRSV-rev and pMD2.G
helper plasmids were mixed in a weight ratio of 4:3:2:2, and diluted with an Opti-MEM
medium (purchased from GIBCO) to give a solution A. A PEI diluent was prepared in
a ratio of total plasmids:PEI = 3:1, and diluted with the Opti-MEM medium to obtain
a solution B. The solutions A and B were mixed well and incubated at room temperature
for 15 min.
- (3) The 293T cells were immobilized on a plate, and slowly added with the plasmid-PEI
mixture. The resulting mixture was shaken gently, and cultivated in a CO2 incubator at 37 °C/5% CO2 for 4-6 h. After incubation, the medium was replaced with a fresh DMEM medium containing
10% FBS.
- (4) After 48 h and 96 h of transfection, the culture supernatant containing viruses
was collected and centrifuged at 3000 rpm at 4 °C for 5 min. The supernatant was filtered
through a 0.45 µm filter, mixed with PEG8000/NaCl in a volume ratio of 4:1, incubated
at 4 °C for 2-3 h, and centrifuged at a high speed for 30 min. The supernatant was
discarded and the precipitate was resuspended with precooled T cell medium X-VIVO
15 (Lonza, 04-418Q) or PBS to give a virus concentrate which was stored at -80 °C
for later use.
6.4 Lentivirus titer assay
[0091] In the example, the biological activity titer of lentivirus was determined by infecting
cells. The 293T cells were used for lentivirus activity assay, and 1 × 10
5 cells were inoculated to each well of a 24-well culture plate. 1 mL of fresh DMEM
medium containing 10% FBS was added to each well. The mixture was diluted to a final
concentration of 6 µg/mL with transfection additive Polybrene. The lentivirus concentrate
was serially diluted by 3 × to the 5th concentration, added at 1 µL/well in duplicate,
and mixed well. The cells were incubated in a CO
2 incubator at 37 °C/5% CO
2 for 24 h. After 24 h, the cells were digested, and the positive rate of protein expression
of CAR or EGFRt was detected by a flow cytometer using an anti-human IgG(Fab)
2 (Jackson ImmunoResearch, 109-065-006) or anti-human EGFRt (Biolegend, 352904) flow
dye. The titer was calculated by the following formula: lentivirus activity titer
(TU/mL) = positive rate × dilution factor × 100 × 10
5. The activity titers of lentivirus concentrates of the above CAR (CN01, CN02, CN03
and CN04) packaged by PEI transfection were greater than 1 × 10
8TU/mL (FIG. 12).
6.5 Preparation of T lymphocytes
[0092] Peripheral blood mononuclear cells (PBMCs) purchased from AllCells were marked with
microbeads through a CD3 MicroBeads human-lyophilized Kit (purchased from Miltenyi
Biotech). CD3+ T lymphocytes with high purity were selected, with a proportion of
CD3 positive T cells over 95%. The purified T cells were activated and proliferated
using a human CD3CD28 T cell activator (Dynabeads Human T-Activator CD3/CD28, Thermo
Fisher, 11132D).
6.6 Lentivirus-transduced T lymphocytes
[0093] CAR-T cells were obtained by transducing T cells with the lentivirus prepared in
Section 6.3. After stimulated and activated for 24-48 h, T lymphocytes from Section
6.5 were observed using microscopy for their activation. Activated T lymphocytes are
larger in volume with elongated or irregular shape. The activated T lymphocytes were
collected, centrifuged and resuspended in a T cell medium X-VIVO 15 (Lonza, 04-418Q)
with a final concentration of 10 ng/mL IL-7 and 5 ng/mL IL-15 and a final volume of
1 mL, and added to a 12-well culture plate. The lentivirus was diluted to MOI = 3-5
with the same medium and mixed with 1 × 10
6 activated T lymphocytes for infection. The mixture was incubated overnight on a 24-well
plate in an incubator at 37 °C/5% CO
2. The next day, the cells were centrifuged again and the medium was refreshed. The
cell density was measured every 2 days thereafter, and the cells were further expanded
with the cell density controlled at NMT 2 × 10
6 cells/mL. After the T cells were co-incubated with the lentivirus for 48-72 h, the
expression of different chimeric antigen receptors was determined by flow cytometry.
With non-transduced T lymphocytes as negative control, the positive rates of T lymphocytes
expressing different chimeric antigen receptors are shown in Table 16 (FIG. 13).
Table 16. Positive rates of T lymphocytes expressing different chimeric antigen receptors
Cells transfected with the following CAR |
Positive rate of CAR |
CN01 |
30.5% |
CN02 |
19.1% |
CN03 |
24.3% |
CN04 |
13.2% |
[0094] After being infected with lentiviruses packaging different chimeric antigen receptors,
T lymphocytes were cultivated for about 9 days, reaching about 300× expansion, which
indicated that T lymphocytes expressing different chimeric antigen receptors could
be expanded
in vitro to a certain extent, providing a guarantee for subsequent
in vitro functional studies and pharmacodynamic studies in animals.
6.7 In vitro toxicity assay
6.7.1 Target specificity assay
[0095] Claudin-18 has two splicing variants,
i.e., Claudin 18.1 and Claudin 18.2, with only eight amino acid alterations in sequence.
Claudin 18.1 is selectively expressed in normal lung cells, while Claudin 18.2 is
highly restricted in normal cells, but is frequently ectopically activated and overexpressed
in multiple tumors (e.g., gastric cancer, lung cancer, pancreatic cancer). In the
example, with 293T cells (purchased from KYinno, KC-0990/KC-0986) overexpressing Claudin18.1
protein and Claudin18.2 protein being the target cells and prepared Claudin18.2 CAR-T
with different scFvs being the effector cell, a co-incubation system of CAR-T cells
and target cells was established using 293T cells, 293T cells overexpressing Claudin18.2
protein and 293T cells overexpressing Claudin18.1 protein in different E:T (effector
cells:target cells) ratios. The specific response of CAR-T to the two proteins was
evaluated by measuring the lysis rate of tumor cells. The results of
in vitro assay (FIG. 14) demonstrated that, with a fixed number of tumor cells, the efficiency
of killing tumor cells ranged from 20%-50% at 24 h (Table 17) when the prepared Claudin18.2
CAR-T with different scFvs (CN01, CN02, CN03, CN04) was co-incubated with 293T-hClaudin18.2
cells at E:T ratios of 1:1 and 3:1. When co-incubated with 293T-hClaudin18.1 cells
at E:T ratios of 1:1 and 3:1, the prepared Claudin18.2 CAR-T cells with different
scFvs were significantly different in specific lysis ability against 293T-hClaudin18.1
cells, among which the CN02 CAR-T had no significant specific lysis ability against
293T-hClaudin18.1 cells, while the CN01, CN03 and CN04 CAR-T had different degrees
of specific lysis ability against 293T-hClaudin18.1 cells. The specific response of
CAR-T cells was also evaluated by measuring the content of cytokines (INF-gamma) secreted
into the culture supernatant. The difference in release of IFN-gamma cytokines in
supernatant of the Claudin18.2 CAR-T (CN01, CN02, CN03, CN04) with different scFvs
and the 293T-hClaudin18.1 cells was consistent with the results of killing assay (FIG.
15 and Table 18). The IFN-gamma cytokines in 293T-hClaudin18.1 cells co-incubation
supernatant in CN02 group was significantly lower than that in CN01, CN03 and CN04
groups.
[0096] Specific lysis assay: an LDH Release Assay Kit (Dojindo, CK12) was used for assay,
which is an INT chromogenic reaction catalyzed by diaphorase, and measures the activity
of LDH released during cytotoxicity via colorimetry. Damage to the cell membrane structure
caused by cell apoptosis or necrosis will lead to release of enzymes in cytoplasm
into the cultures, including lactate dehydrogenase (LDH) with relatively stable enzymatic
activity. The cytotoxicity can be quantitatively analyzed by activity assay of LDH
released from lysed cells into the cultures. LDH release is considered as an important
indicator of cell membrane integrity and is widely used for cytotoxicity assay.
[0097] Cytokine assay: Human IFN-gamma ELISA kit (R&D Systems, SIF50) was used for measuring
cytokines, which is based on the immobilization of an antigen or antibody and enzymatic
labeling of the antigen or antibody. The antigen or antibody that binds to the surface
of a solid carrier retains the immunological activity, while the enzyme labeled antigen
or antibody retains both immunological activity and enzymatic activity. During the
assay, the test substance (the antigen or antibody) in the sample are bound to the
immobilized antibody or antigen. Non-binding substances are removed by washing, and
the enzyme-labeled antigen or antibody is added. In this case, the amount of enzyme
immobilized is associated with the amount of the test substance in the sample. After
a substrate that reacts with the enzyme is added for color development, the content
of the test substance in the sample could be judged by the color for qualitative or
quantitative analysis.
Table 17. Specific lysis assay of Claudin18.2 CAR-T cells with different scFvs
in vitro
Specific lysis rate (E:T=1:1) |
T |
CN01 |
CN02 |
CN03 |
CN04 |
293T |
1.60% |
5.90% |
7.25% |
9.18% |
3.96% |
293T-hClaudin18.1 |
2.42% |
17.60% |
5.75% |
34.85% |
15.25% |
293T-hClaudin18.2 |
0.77% |
28.31% |
39.68% |
51.53% |
23.78% |
Specific lysis rate (E:T=3:1) |
T |
CN01 |
CN02 |
CN03 |
CN04 |
293T |
1.10% |
14.91% |
19.21% |
23.08% |
8.93% |
293T-hClaudin18.1 |
2.72% |
30.79% |
11.98% |
44.53% |
32.91% |
293T-hClaudin18.2 |
-0.49% |
58.02% |
55.86% |
65.62% |
49.72% |
Table 18. Release of IFN-gamma cytokines in supernatant of co-incubated Claudin18.2
CAR-T cells with different scFvs and different 293T cells
IFN -gamma pg/mL (E:T=1:1) |
T |
CN01 |
CN02 |
CN03 |
CN04 |
293T |
0.00 |
0.00 |
0.00 |
578.76 |
0.00 |
293T-hClaudin18.1 |
0.00 |
1059.53 |
0.00 |
7229.32 |
3291.64 |
293T-hClaudin18.2 |
0.00 |
3818.19 |
7091.96 |
11235.69 |
7961.92 |
IFN -gamma pg/mL (E:T=3:1) |
T |
CN01 |
CN02 |
CN03 |
CN04 |
293T |
0.00 |
17.87 |
0.00 |
899.27 |
0.00 |
293T-hClaudin18.1 |
0.00 |
1952.37 |
0.00 |
10411.52 |
4619.46 |
293T-hClaudin18.2 |
0.00 |
4791.17 |
10171.14 |
14188.95 |
8889.10 |
6.7.2 Target toxicity assay
[0098] In the example, an
in vitro pharmacodynamic test was established by simulating the mechanism of action (MOA)
of the product. The inventor constructed a plasmid overexpressing Claudin18.2 protein
with a plvx vector, and prepared a lentivirus. Gastric cancer cells NUGC4 and AGS
were infected with the lentivirus, and NUGC4 cells and AGS cells with high expression
of Claudin18.2 protein were obtained through subsequent screening of positive cells
as target cells for functional verification of CAR-T cells. Claudin18.2 CAR-T cells
with different scFvs prepared above were used as effector cells. A co-incubation system
of CAR-T cells and target tumor cells was established in different E:T (effector cells:target
cells) ratios. The biological efficacy of the CAR-T cells was evaluated by measuring
the lysis rate of tumor cells, with a co-incubation system of non-transduced T cells
and tumor cells being the control.
[0099] The results of
in vitro assay (FIG. 16 and Table 19) demonstrated that the efficiency of killing tumor cells
was excellent at 24 h when CAR-T cells were co-incubated with Claudin18.2-positive
tumor cells (AGS-claudin18.2 and NUGC-4), which was significantly higher than that
of T cells, while the killing effect on the Claudin18.2-negative cell line AGS was
mild. Also, the biological efficacy of CAR-T cells was evaluated by measuring the
content of cytokines (INF-gamma) secreted into the culture supernatant. After Claudin18.2
CAR-T cells were co-incubated with Claudin18.2-positive tumor cells (AGS-claudin18.2
and NUGC-4), the expression of the IFN-gamma cytokines was significantly higher than
that of the T cell group (FIG. 17, Table 20).
Table 19. Specific lysis assay of Claudin18.2 CAR-T cells with different scFvs against
Claudin18.2-positive tumor cells
Specific lysis rate (E:T=1:1) |
T |
CN01 |
CN02 |
CN03 |
CN04 |
AGS |
2.01% |
8.83% |
6.32% |
10.89% |
-1.12% |
AGS-claudin18.2 |
7.61% |
43.94% |
41.18% |
47.84% |
37.02% |
NUGC-4 |
5.05% |
58.25% |
52.22% |
68.23% |
57.64% |
Specific lysis rate (E:T=3:1) |
T |
CN01 |
CN02 |
CN03 |
CN04 |
AGS |
3.11% |
17.53% |
14.67% |
20.50% |
13.18% |
AGS-claudin18.2 |
15.92% |
44.20% |
43.86% |
58.56% |
41.87% |
NUGC-4 |
5.17% |
64.78% |
59.11% |
66.50% |
66.38% |
Table 20. Release of IFN-gamma cytokines in supernatant of co-incubated Claudin18.2
CAR-T cells with different scFvs and Claudin18.2-positive tumor cells
IFN -gamma pg/mL (E:T=1:1) |
T |
CN01 |
CN02 |
CN03 |
CN04 |
AGS |
6.42 |
475.74 |
235.36 |
842.04 |
304.04 |
AGS-claudin18.2 |
0.00 |
2787.98 |
4470.66 |
7732.98 |
4962.87 |
NUGC-4 |
0.00 |
8580.04 |
7401.03 |
13731.08 |
9266.85 |
IFN -gamma pg/mL (E:T=3:1) |
T |
CN01 |
CN02 |
CN03 |
CN04 |
AGS |
63.66 |
979.40 |
349.83 |
613.10 |
0.00 |
AGS-claudin18.2 |
0.00 |
3955.55 |
8145.06 |
11968.28 |
6130.44 |
NUGC-4 |
0.00 |
6382.26 |
4745.38 |
11624.88 |
7206.43 |
[0100] The
in vitro cytotoxicity assay shows that all the T lymphocytes expressing different chimeric
antigen receptors have good killing ability on Claudin18.2-positive tumor cells, which
provides a basis for pharmacodynamic studies in animals.
6.8 Studies in animals
[0101] In the example, a pharmacodynamic model of immunodeficient mouse bearing gastric
cancer tumor was established. Based on
in vitro studies, each of the female NOG mice (purchased from Charles River) was grafted with
1 × 10
7 NUGC4-Claudin18.2 cells on the back. The mice were administered on Day 11 after grafting
(the tumor volume was about 80-100 mm
3). The vehicle control group was administered with 0.9% normal saline, the Mock-T
(T cells not transfected with plasmid) group was administered with 1 × 10
7 cells, and CN02 low-dose and high-dose groups (positive cells) were administered
with 5.00 × 10
6 and 1.00 × 10
7 cells respectively. The dose volume was 100 µL. 6 animals were allocated in each
group. Tumors were measured twice a week after administration. The tumor growth curve
was plotted, TGI and T/C were calculated, and all tumors were photographed at the
end of the study. Blood was sampled before CAR-T administration (Day -2), on Days
2, 9 and 28 after administration, and the vector copy number (VCN) of CAR in peripheral
blood of mice was measured by qPCR, so as to confirm the expansion of CART cells.
The results showed that within 36 days after CAR-T administration, the efficacy of
both treatment group was significant. In the 6 mice in the Claudin18.2 CAR-T (CN02)
low-dose group, the tumor regressed completely (6/6), and in 5 mice in the high-dose
group, the tumor regressed completely (5/6) (FIG. 18).
- (1) Body weight: compared with the vehicle control and Mock groups, the Claudin18.2
CAR-T (CN02) low-dose and high-dose groups had no significant difference in body weight
(FIG. 19).
- (2) Tumor growth inhibition (TGI): 14 days after administration, the TGI of the Claudin18.2
CAR-T (CN02) low-dose and high-dose groups was 100% and 88.33% respectively (FIG.
20).
- (3) Death rate: by 31 days after administration, one animal was found dead in the
vehicle control group, and no death was observed in the Claudin18.2 CAR-T (CN02) low-dose
and high-dose groups (FIG. 21).
Example 7. Preparation of CAR-T cells by non-viral method
7.1 Construction of non-viral PiggyBac(PB) transposon vector
[0102] In the example, with a pBluescirpt vector (synthesized by General BIOL) as the backbone,
a gene insulator sequence cHS4 was found and placed at both termini of a polyclonal
site, 5'ITR and 3'ITR sequences of PB transposon were found and constructed inside
the cHS4 sequence of the vector, an EF1a promoter was inserted at the 5' terminus
inside the ITR, and a polyA signal was inserted at the 3' terminus. A polyclonal sequence
was retained in the middle, into which a CAR-P2A-EGFRt sequence was inserted to form
a PB CN02 CAR plasmid structure, as shown in FIG. 24.
7.2 Preparation of CAR-T cells by non-viral PB transposon vector
[0103] 7.2.1 Peripheral blood mononuclear cells (PBMCs) purchased from AllCells were marked
with microbeads through a CD3 MicroBeads human-lyophilized Kit (purchased from Miltenyi
Biotech). CD3+ T lymphocytes with high purity were selected, with a proportion of
CD3 positive T cells over 95%. The purified T cells were activated and proliferated
using a human CD3CD28 T cell activator (Dynabeads Human T-Activator CD3/CD28, Thermo
Fisher, 11132D).
[0104] 7.2.2 Electroporation was performed on Day 3 after stimulation. The cells for electroporation
were resuspended using a pipette and counted. 5 × 10
6 cells were used for each electroporation. The 5 × 10
6 cells were diluted with DPBS (GIBCO, 14190-144) to 4 mL, and centrifuged at 300 g
at room temperature for 10 min. The supernatant was discarded, and the cells were
resuspended and washed with 5 mL of DPBS, and centrifuged at 300 g at room temperature
for 10 min and then the supernatant was discarded. The cells were then resuspended
in 100 µL of electroporation buffer Entranster-E (Engreen, 98668-20), and the cell
suspension was transferred to a 1.5-mL centrifuge tube.
[0105] The components in Table 21 were added to the centrifuge tube and mixed well.
Table 21. Electroporation system
Component |
Volume (µL) |
PB CN02 CAR plasmid (1 µg/µL) |
5 |
PB transposase plasmid (1 µg/µL) |
5 |
Cell suspension |
100 |
Total |
110 |
[0106] Electroporation was performed using an electroporation instrument manufactured by
Lonza. The cell/plasmid suspension was quickly transferred to the cuvette, and the
cuvette was tapped to allow the cell suspension to fully form a balanced liquid level
in the cuvette. The program EO115 was used for electroporation. The cuvette was taken
out carefully after electroporation. Then 500 µL of preheated T cell medium X-VIVO
15 (Lonza, 04-418Q) was added and equilibrated in an incubator at 37 °C for 5 min,
and the cells were resuspended using a microporous loading tip by blowing 2-3 times.
The cells were transferred to a 12-well plate containing 2 mL of preheated medium
and incubated at 37 °C. The medium was refreshed 4-6 h after electroporation to improve
the viability. The supernatant was discarded, and preheated fresh medium was added.
The cells were incubated in an incubator at 37 °C/5% CO
2 for 48h before the test.
[0107] Meanwhile, a CAR-T control group using lentiviruses was set. The preparation method
can be seen in Section 6.6.
[0108] 48-72 h after electroporation, the expression of chimeric antigen receptors was determined
by flow cytometry using an anti-human IgG(Fab)
2 antibody, with non-transduced T lymphocytes as negative control. The positive rates
of T lymphocytes expressing different chimeric antigen receptors are shown in Table
22 (FIG. 22).
Table 22. Positive rates of T lymphocytes expressing different chimeric antigen receptors
Cell |
Positive rate of CAR, % |
Non-transduced T cell |
0.56 |
Non-viral PB CN02 CAR-T |
35.4 |
Lentiviral Lenti CN02 CAR-T |
38 |
7.3 Target toxicity assay
[0109] In the example, an
in vitro pharmacodynamic test was conducted by simulating the mechanism of action (MOA) of
the product. The constructed gastric cancer cells NUGC4 and AGS with high expression
of Claudin18.2 were used as target cells, and CN02 CAR-T cells prepared using the
above non-viral (PB) and CN02 CAR-T cells prepared using lentiviruses (lenti) were
used as effector cells. A co-incubation system of CAR-T cells and target tumor cells
was established in different E:T (effector cells:target cells) ratios. The biological
efficacy of the CAR-T cells was evaluated by measuring the lysis rate of tumor cells,
with a co-incubation system of non-transduced T cells and tumor cells being the control.